Research in Progress: This project is in collaboration with: Ambika Bumb and Martin W. Brechbiel Radiation Oncology Branch, National Cancer Institute, National Institutes of Health and Fedor Jelezko Physikalisches Institute, University of Stuttgart Currently, there are two main projects: The first project involves the use of single-molecule techniques to measure the optical properties and characteristics of multimodal in vivo imaging probes. One class of particles consists of an iron core in a silica shell in which organic fluorophores are encapsulated during the synthesis process. Because of the complex nature of the particles, it has proved difficult to reliably determine the average number of incorporated fluorophores and the fraction of particles that are labeled using traditional ensemble measurement techniques. In collaboration with Ambika Bumb and Martin Brechbiel of the Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, we used the custom built prism-based total internal reflection fluorescence (TIRF) microscope and single-molecule imaging capabilities in our lab to measure the fluorescence properties of these synthesized particles. By measuring the fluorescence from single particles as a function of time, we are able to directly observe the photo-bleaching of individual dyes in the particles. The number of photo-bleaching steps that reduce the fluorescence to background levels is indicative of the number of dyes in each particle. The magnitude of each discrete decrease in intensity is indicative of the brightness of the individual dyes, whereas the time between photo-bleaching steps directly provides the photo-bleaching rate, or the photo-stability of the dye. The wide-field single-molecule TIRF set-up allowed the collection of thousands of individual fluorescence traces, providing excellent statistical samples. In a proof-of-principle experiment, we were able to determine the average number of dyes per particle for 15 nm iron core silica particles embedded with either Alexa 555 or Cy 5.5 dyes. Further analysis of the fluorescence traces revealed that encapsulation of the dye increased its fluorescence intensity and increased its photo-stability as evidenced by brighter emission and longer bleaching times as compared with free dye. The distribution of the number of dyes per particle was well described by a Poisson distribution. This allowed us to infer the fraction of particles that were labeled, which is difficult to ascertain by ensemble methods. We anticipate that this relatively simple, robust and rapid technique that requires trivial amounts of material will be of general interest to the nanoparticle and molecular imaging fields. In ongoing experiments we are testing the statistical models of labeling by directly determining the fraction of fluorescent particles, through a combination of single particle imaging and TIRF microscopy. In a second project we are collaborating with Ambika Bumb and Martin Brechbiel of the Radiation Oncology Branch, National Cancer Institute, National Institutes of Health and Fedor Jelezko of the Physikalisches Institute University of Stuttgart, Stuttgart Germany on functionalizing and characterizing nitrogen vacancy fluorescent nanodiamonds (FNDs) for use as multi-modal imaging probes. These are attractive fluorescence particles for in vivo and in vitro tracking and imaging studies as they are bright, non-blinking fluorophores that are excited in the green (532 nm) and emit in the far red spectrum (700 nm), which has superior tissue penetration and signal-to-noise characteristics compared with shorter wavelengths in biological samples. Moreover, diamond is inert and the fluorescence arises from the nitrogen vacancy so the core particle contains no organic dyes or other potentially toxic material that would be problematic for in vivo applications. Remarkably, the FNDs can be as small as 5 nm, which is also advantageous for biocompatibility and clearing. The initial goal of the project is to establish protocols to functionalize 5-10 nm FNDs and attach gadolinium chelates as MR contrast agents. This will be followed by in vivo tracking and biodistribution and clearing studies of the functionalized and labeled FNDs to establish feasibility and biocompatibility in an in vivo model. In parallel we will optimize the functionalization to facilitate in vitro protein labeling for single-molecule fluorescence tracking applications.